This application is based upon and claims the benefit of priority from the prior Japanese Patent Application No. 2000-087302, filed Mar. 27, 2000, the entire contents of which are incorporated herein by reference.
The present invention relates to a method of preparing a guanosine-group compound, which is used as a raw material of an anti-virus agent, antisense medicine and the like, and a method of preparing a glyoxal-guanosine-group compound as an intermediate of the guanosine-group compound.
Guanosine or 2xe2x80x2-deoxyguanosine is industrially produced mainly by extraction/separation of hydrolysate of DNA (deoxyribonucleic acid) or RNA (ribonucleic acid), because the yield is extremely low when guanosine or 2xe2x80x2-deoxyguanosine is chemically synthesized. However, the hydrolysate of DNA contains 2xe2x80x2-deoxyadenosine, 2xe2x80x2-deoxycytidine and thymidine other than the targeted 2xe2x80x2-deoxyguanosine. The hydrolysate of RNA contains adenosine, cytidine and uridine other than the targeted guanosine. Accordingly, in order to collect only guanosine or 2xe2x80x2-deoxyguanosine, it is necessary to perform complicated processes of extraction/separation, thereby inevitably raising the production cost.
On the other hand, a method has been reported in which nucleoside (or deoxynucleoside) and nucleic acid base, which are the raw materials, are subjected to a base-exchange reaction by nucleoside phosphorylase, whereby the aimed nucleoside (or the aimed deoxynucleoside) is obtained (Hori, N., Watanabe, M., Yamazaki, Y., Mikami, Y., Agric. Biol. Chem., 53, 197-202 (1989)). By this method, adenosine and 2xe2x80x2-deoxyadenosine can be easily prepared (Jpn. Pat. Appln. KOKAI Publication No. 11-46790 xe2x80x9cMethod of preparing a purine nucleoside compoundxe2x80x9d). In order to obtain guanosine and 2xe2x80x2-deoxyguanosine by this method of enzymatic synthesis reaction, guanine must be soluble to water at least in a range of pH where the enzymes necessary for the reaction can function. In general, it is considered that the solubility of the reaction substrate is preferably at least equal to the concentration of Michaelis constant (Km) of the enzyme or higher, in order to carry out the enzyme reaction smoothly. However, as the solubility of guanine to water does not exceed a few ppm, the above method of enzymatic synthesis reaction cannot be employed in practice. Due to such an impasse-like situation, it has been demanded to develop a method of efficiently preparing guanosine or 2xe2x80x2-deoxyguanosine.
An object of the present invention is to provide a method of efficiently preparing guanosine or 2xe2x80x2-deoxyguanosine (i.e., a guanosine-group compound) at a high yield, as well as a method of efficiently preparing a glyoxal-guanosine or glyoxal-2xe2x80x2-deoxyguanosine (i.e., glyoxal-guanosine-group compound) as an intermediate of the guanosine-group compound at a high yield.
The inventors of the present invention have assiduously studied for solving the aforementioned problems. As a result, the inventors have discovered the following 1)-6).
1) When guanine, whose solubility to water does not exceed a few ppm, is reacted with glyoxal, glyoxal-guanine represented by the following formula (1) is obtained, and glyoxal-guanine is soluble to water. 
(Glyoxal-guanine is also referred to as 6,7-dihydro-6,7-dihydroxyimidazo[1,2-a]purine-9(3H)-one.)
2) When microorganism itself which contains purine nucleoside phosphorylase (EC 2.4.2.1) or this enzyme derived from the microorganism are applied, for a reaction, to glyoxal-guanine and any one of ribose-1-phosphate and 2-deoxyribose-1-phosphate, a glyoxal-guanosine-group compound (i.e., glyoxal-guanosine or glyoxal-2xe2x80x2-deoxyguanosine) represented by the following formula (2) is obtained. Alternatively, when microorganism itself which contains purine nucleoside phosphorylase (EC 2.4.2.1) and pyrimidine nucleoside phosphorylase (EC 2.4.2.2) or these enzymes derived from the microorganism are applied, for a reaction, to glyoxal-guanine and any one selected from the group consisting of uridine, 2xe2x80x2-deoxyuridine and thymidine under the presence of phosphate ion, a glyoxal-guanosine-group compound (i.e., glyoxal-guanosine or glyoxal-2xe2x80x2-deoxyguanosine) represented by the following formula (2) is obtained. In other words, glyoxal-guanine can be a substrate of purine nucleoside phosphorylase. 
(wherein R represents a hydrogen atom or a hydroxyl group.)
(Glyoxal-guanosine is also referred to as 3-(xcex2-D-erythropentofuranosyl)-6,7-dihydro-6,7-dihydroxyimidazo[1,2-a]purine-9(3H)-one, and glyoxal-2xe2x80x2-deoxyguanosine is also referred to as 3-(2-deoxy-xcex2-D-erythropentofuranosyl)-6,7-dihydro-6,7-dihydroxyimidazo[1,2-a]purine-9(3H)-one.)
3) In the method of preparing a glyoxal-guanosine-group compound described in 2) above, when at least one compound selected from the group consisting of glycine, iminodiacetic acid, nitrilotriacetic acid, ethylenediaminetetraacetic acid (which will be referred to as xe2x80x9cEDTAxe2x80x9d hereinafter), ethylene glycol bis(xcex2-aminoethyl ether)-N,N,Nxe2x80x2,Nxe2x80x2-tetraacetic acid (which will be referred to as xe2x80x9cEGTAxe2x80x9d hereinafter) and salts of these substances is added, or the above at least one compound is added in combination with boric acid or a salt thereof, the reaction yield is significantly improved. The above compound is added as a stabilizer of ribose-1-phosphate or 2-deoxyribose-1-phosphate, which is the substrate or an intermediate produced during base-exchange reaction.
4) As a principle, nucleoside phosphorylase derived from various organism can be efficient as the enzyme to be used in the preparation method of 2) above. However, the enzyme derived from microorganism of Bacillus genus, Escherichia genus or Klebsiella genus is suitable, and the thermotolerant enzyme derived from Bacillus stearothermophilus JTS 859 (FERM BP-6885) is especially preferable.
5) When an alkali is applied, for a reaction, to the glyoxal-guanosine-group compound obtained by the method of 2) above, the glyoxal portion of the glyoxal-guanosine-group compound is easily released, whereby a guanosine-group compound (i.e., guanosine or 2xe2x80x2-deoxyguanosine) can be obtained.
6) The preparation processes of the aforementioned 1)-5) can be carried out by one-pot reaction.
On the basis of the aforementioned discoveries, the present invention has been completed.
In summary, the present invention relates to the following methods (1)-(16).
(1) A method of preparing a glyoxal-guanosine-group compound represented by the formula (2): 
wherein R represents a hydrogen atom or a hydroxyl group, which comprises the step of:
reacting glyoxal-guanine represented by the formula (1): 
xe2x80x83with ribose-1-phosphate or 2-deoxyribose-1-phosphate in the presence of purine nucleoside phosphorylase, thereby obtaining a glyoxal-guanosine or glyoxal-2xe2x80x2-deoxyguanosine.
(2) A method of preparing a glyoxal-guanosine-group compound represented by the formula (2): 
wherein R represents a hydrogen atom or a hydroxyl group, which comprises the step of:
reacting glyoxal-guanine represented by the formula (1): 
xe2x80x83with any one selected from the group consisting of uridine, 2xe2x80x2-deoxyuridine and thymidine, together with phosphate ion, in the presence of purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase, thereby obtaining a glyoxal-guanosine or glyoxal-2xe2x80x2-deoxyguanosine.
(3) A method of preparing a guanosine-group compound, which comprises the steps of:
reacting glyoxal-guanine represented by the formula (1): 
xe2x80x83with ribose-1-phosphate or 2-deoxyribose-1-phosphate in the presence of purine nucleoside phosphorylase, thereby obtaining a compound represented by the formula (2): 
xe2x80x83wherein R represents a hydrogen atom or a hydroxyl group; and
decomposing, by alkali, the compound represented by the formula (2), thereby obtaining guanosine or 2xe2x80x2-deoxyguanosine.
(4) A method of preparing a guanosine-group compound, which comprises the steps of:
reacting glyoxal-guanine represented by the formula (1): 
xe2x80x83with any one selected from the group consisting of uridine, 2xe2x80x2-deoxyuridine and thymidine, together with phosphate ion, in the presence of purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase, thereby obtaining a compound represented by the formula (2): 
xe2x80x83wherein R represents a hydrogen atom or a hydroxyl group; and
decomposing, by alkali, the compound represented by the formula (2), thereby obtaining guanosine or 2xe2x80x2-deoxyguanosine.
(5) A method of preparing a glyoxal-guanosine-group compound described in the aforementioned (1), wherein, as purine nucleoside phosphorylase, a microorganism itself which contains the enzyme or the enzyme derived from the microorganism is used.
(6) A method of preparing a glyoxal-guanosine-group compound described in the aforementioned (2), wherein, as purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase, a microorganism itself which contains the enzymes or the enzymes derived from the microorganism are used.
(7) A method of preparing a guanosine-group compound described in the aforementioned (3), wherein, as purine nucleoside phosphorylase, a microorganism itself which contains the enzyme or the enzyme derived from the microorganism is used.
(8) A method of preparing a guanosine-group compound described in the aforementioned (4), wherein, as purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase, a microorganism itself which contains the enzymes or the enzymes derived from the microorganism are used.
(9) A method of preparing a glyoxal-guanosine-group compound described in the aforementioned (5) or (6), wherein the microorganism belongs to Bacillus genus, Escherichia genus or Klebsiella genus.
(10) A method of preparing a guanosine-group compound described in the aforementioned (7) or (8), wherein the microorganism belongs to Bacillus genus, Escherichia genus or Klebsiella genus.
(11) A method of preparing a glyoxal-guanosine-group compound described in any one of the aforementioned (5), (6) and (9), wherein the microorganism is Bacillus stearothermophilus JTS 859 (FERM BP-6885), Escherichia coli IFO 3301, Escherichia coli IFO 13168, or Klebsiella pneumoniae IFO 3321.
(12) A method of preparing a guanosine-group compound described in any one of the aforementioned (7), (8) and (10), wherein the microorganism is Bacillus stearothermophilus JTS 859 (FERM BP-6885), Escherichia coli IFO 3301, Escherichia coli IFO 13168, or Klebsiella pneumoniae IFO 3321.
(13) A method of preparing a glyoxal-guanosine-group compound described in any one of the aforementioned (1), (2), (5), (6), (9) and (11), wherein at least one compound selected from the group consisting of glycine, iminodiacetic acid, nitrilotriacetic acid, ethylenediaminetetraacetic acid, ethylene glycol bis(xcex2-aminoethyl ether)-N,N,Nxe2x80x2,Nxe2x80x2-tetraacetic acid and salts thereof is added, or the above at least one compound is added in combination with boric acid or a salt thereof.
(14) A method of preparing a guanosine-group compound described in any one of the aforementioned (3), (4), (7), (8), (10) and (12), wherein at least one compound selected from the group consisting of glycine, iminodiacetic acid, nitrilotriacetic acid, ethylenediaminetetraacetic acid, ethylene glycol bis(xcex2-aminoethyl ether)-N,N,Nxe2x80x2,Nxe2x80x2-tetraacetic acid and salts thereof is added, or the above at least one compound is added in combination with boric acid or a salt thereof.
(15) A method of preparing a glyoxal-guanosine-group compound described in any one of the aforementioned (1), (2), (5), (6), (9), (11) and (13), which is performed by one-pot reaction.
(16) A method of preparing a guanosine-group compound described in any one of the aforementioned (3), (4), (7), (8), (10), (12) and (14), which is performed by one-pot reaction.
Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.
The present invention will be described in detail hereinafter.
 less than Raw Material greater than 
Glyoxal-guanine (i.e., 6,7-dihydro-6,7-dihydroxyimidazo[1,2-a]purine-9(3H)-one) to be used as a raw material in the present invention can be prepared at a high yield by adding guanine to a glyoxal aqueous solution and stirring the mixture with heating at a high temperature (preferably at 50-75xc2x0 C.) for about 15-20 hours. The solubility of glyoxal-guanine to water increases as the temperature rises. After the completion of the reaction, glyoxal-guanine is crystallized by cooling and can be obtained as a white solid by filtering.
However, when one-pot reaction is carried out by continuously adding nucleoside phosphorylase and either ribose donor or 2-deoxyribose donor, the solution in which glyoxal-guanine has been obtained may be used, as it is, as a raw material solution.
In the present invention, ribose-1-phosphate and uridine used as the ribose donor, and 2-deoxyribose-1-phosphate, 2xe2x80x2-deoxyuridine and thymidine used as the 2-deoxyribose donor are commercially available by, for example, Sigma Aldrich Japan Co.
 less than Synthesis Conditions of a Glyoxal-guanosine-group Compound by the Enzyme Reaction greater than 
The ribose donor or the 2-deoxyribose donor (which will be referred to as xe2x80x9cthe ribose-group donorxe2x80x9d hereinafter) and glyoxal-guanine are added to a phosphate buffer solution (pH 6-8, preferably pH 7). Microorganism itself or the enzymes derived from the microorganism, which produces purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase (or only purine nucleoside phosphorylase), is added to the above phosphate buffer mixture. The resultant reaction mixture is stirred at the optimum temperature for the enzyme for 24-60 hours, resulting in reactions as represented by xe2x80x9cReaction formula 1 and Reaction formula 2xe2x80x9d or xe2x80x9cReaction formula 3 and Reaction formula 4xe2x80x9d. Glyoxal-2xe2x80x2-deoxyguanosine is obtained by xe2x80x9cReaction formula 1 and Reaction formula 2xe2x80x9d. Glyoxal-guanosine is obtained by xe2x80x9cReaction formula 3 and Reaction formula 4xe2x80x9d. In Reaction formula 1 and Reaction formula 3 as the former half part, pyrimidine nucleoside phosphorylase is used as the enzyme in the reaction. In Reaction formula 2 and Reaction formula 4 as the latter half part, purine nucleoside phosphorylase is used as the enzyme in the reaction. With respect to the optimum temperature for the enzyme, the temperature may be in the range of approximately 40-70xc2x0 C. in the case of the enzyme derived from Bacillus stearothermophilus JTS 859 (FERM BP-6885).
In the enzyme reaction of the present invention, purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase (or only purine nucleoside phosphorylase) are used. When pyrimidine nucleoside (uridine, 2xe2x80x2-deoxyuridine, or thymidine) is used as the ribose-group donor, pyrimidine nucleoside phosphorylase is essentially required for producing ribose-1-phosphate or 2-deoxyribose-1-phosphate in xe2x80x9cReaction formula 1xe2x80x9d or xe2x80x9cReaction formula 3xe2x80x9d, as the former half reaction. On the other hand, when ribose-1-phosphate or 2-deoxyribose-1-phosphate is used as the ribose-group donor from the start of the reaction, the reaction represented by xe2x80x9cReaction formula 1xe2x80x9d or xe2x80x9cReaction formula 3xe2x80x9d, is not necessitated, and thus pyrimidine nucleoside phosphorylase is not required. However, in this case, even if pyrimidine nucleoside phosphorylase is present, the overall enzyme reaction will not be adversely affected at all.
In Reaction formulae 1-4, xe2x80x9cPYNPxe2x80x9d represents pyrimidine nucleoside phosphorylase, and xe2x80x9cPUNPxe2x80x9d represents purine nucleoside phosphorylase. 
In the present invention, the initial concentration of the ribose-group donor as the raw material is 5-1000 mM, preferably 10-100 mM. Glyoxal-guanine does not dissolve in a large amount at a time. However, if it is assumed that the initially added amount of glyoxal-guanine were to be completely dissolved at a time, the expressed initial concentration would be 5-1000 mM, preferably 10-100 mM. The initial concentration of phosphate ion of 1-20 mM suffices the purpose.
 less than Stabilizer of Deoxyribose-1-phosphate, etc. greater than 
In the present invention, even when a nucleoside (uridine, 2xe2x80x2-deoxyuridine, thymidine) is used as the raw material, the reaction proceeds by way of ribose-1-phosphate or 2-deoxyribose-1-phosphate as the reaction intermediate, as shown in the aforementioned formulae. These phosphate compounds are unstable by nature and, when being left, naturally decomposed into ribose and phosphate ion or 2-deoxyribose and phosphate ion as time passes. When phosphatase is present in the reaction system, the decomposition is accelerated. If such decomposition occurs, the production efficiency of glyoxal-guanosine-group compound is decreased, because ribose or 2-deoxyribose, which is the product by decomposition of the intermediate, does not serve as the substrate of nucleoside phosphorylase. Therefore, in order to increase the production efficiency, a substance, which stabilizes ribose-1-phosphate or 2-deoxyribose-1-phosphate and inhibits the activity of phosphatase, has been searched in the present invention.
As a result, it has been discovered that addition of at least one compound selected from the group consisting of glycine, iminodiacetic acid, nitrilotriacetic acid, EDTA, EGTA and salts thereof, and more preferably, addition of the above at least one compound in combination with boric acid or a salt thereof is effective. The combination of EDTA or a salt thereof with boric acid or a salt thereof exhibits a significantly excellent effect. The preferable concentration of each composition added is 20-200 mM for boric acid or a salt thereof, and 2-10 mM for the above at least one compound or salts thereof (i.e., glycine, iminodiacetic acid, nitrilotriacetic acid, EDTA, EGTA). Salts of the above compounds and a salt of boric acid used herein are not particularly limited, and any salts can be used. For example, the salts used herein may be salts of alkaline metal such as sodium and potassium, or salts of alkaline earth metal such as calcium and magnesium.
 less than Preparation of a Guanosine-group Compound from a Glyoxal-guanosine-group Compound greater than 
A glyoxal-guanosine-group compound releases, by decomposition, the glyoxal portion thereof in an alkali aqueous solution, thereby obtaining a guanosine-group compound. As the alkali aqueous solution, 0.05-0.5N, preferably 0.1-0.25N sodium hydroxide aqueous solution can be used, for example. This decomposition reaction is accelerated by the increase in pH and temperature. However, it is essential to select a mild condition in which the bonding with ribose-group is not broken. Specifically, a glyoxal-guanosine-group compound is relatively stable in the range of pH 4-8, but is decomposed and releases the glyoxal portion thereof at pH 9 or higher. In conclusion, it is preferable that a glyoxal-guanosine-group compound is decomposed at 60-80xc2x0 C. in the range of pH 9-11 in 1-5 hours duration, to obtain a guanosine-group compound.
 less than Separation and Purification of a Guanosine-group Compound greater than 
Collection of the product from the reaction solution can be performed by ultrafiltration, ion exchange separation, adsorption chromatography, crystallization and the like. The amount of the reaction product can be determined by the HPLC method using a UV detector.
 less than Microorganism and Enzyme greater than 
Purine nucleoside phosphorylase (EC 2.4.2.1) and pyrimidine nucleoside phosphorylase (EC 2.4.2.2) used in the present invention may have any origin as a principle. The enzymes produced by the microorganism described below are preferable. The microorganism to be used in the present invention is not particularly limited to such examples, as long as the microorganism produces the aforementioned enzymes in a significant amount.
However, as shown in the aforementioned Reaction formulae, the present reactions employ nucleoside or 2xe2x80x2-deoxynucleoside as the substrate and thus inevitably produce a phosphate compound as the intermediate. Accordingly, microorganism which shows a strong nucleosidase and/or phosphatase activity cannot be utilized.
Examples of the microorganism which suffices these conditions include those which belong to Bacillus genus, Escherichia genus or Klebsiella genus. More specifically, the examples include Bacillus stearothermophilus JTS 859 (FERM BP-6885), Escherichia coli IFO 3301, Escherichia coli IFO 13168, and Klebsiella pneumoniae IFO 3321.
In the present reaction, the higher the solubility and the dissolving rate of the raw material are, the higher yield and the higher reaction rate are obtained. Accordingly, a relatively high temperature during the reaction is advantageous. Due to this, use of thermophilic bacteria and heat-resistant enzymes is preferable. Among the aforementioned examples of microorganisms, Bacillus stearothermophilus JTS 859 is the most preferable.
xe2x80x9cIFOxe2x80x9d indicates that the microorganisms are preserved in Institute for Fermentation, Osaka (IFO), Feb. 2, 1985, Zyusohonmachi, Yodogawa-ku, Osaka-shi, 532-0024 JAPAN. The xe2x80x9cIFO strainsxe2x80x9d are available to any person, if desired. Bacillus stearothermophilus JTS 859 is under international deposit (the deposit number thereof is FERM BP-6885) in Patent Microorganism Depository, National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology.
The microorganism Bacillus stearothermophilus JTS 859 (FERM BP-6885) was deposited as a national deposit, on Oct. 20, 1987, in the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan (1-3, Higashi 1-Chome, Tsukubashi, Ibaraki-ken, Japan), which is an international deposit authority designated by the BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE. National deposit number P-9666 was assigned to the strain JTS 859. Thereafter, the strain JTS 859 was transferred from the national deposit to an international deposit on Sep. 22, 1999, and international deposit number FERM BP-6885 was assigned to it.
These types of microorganisms are grown on the normal culture medium for bacteria, and show better growth and produce a larger amount of the aforementioned enzyme by adding tryptone, yeast extract, glucose and the like. Further, addition of a nucleic acid compound such as inosine to the culture medium is effective in enhancing the activity of the enzyme. The cultured bacteria itself may be utilized as the crude enzyme. Alternatively, enzyme is obtained from the cultured bacteria by the standard methods (such as destruction by supersonication or milling, centrifugation, ammonium sulfate-fractionation, and membrane separation), and the resultant crude enzyme may also be employed.
The results of studying the bacteriological characteristics of the deposited strain according to xe2x80x9cBergey""s Manual of Determinative Bacteriology, Volume IIxe2x80x9d (1984) are as follows. The experiments were basically carried out by the method described in xe2x80x9cBiseibutsu no Bunrui to Dotei (Classification and Determination of Microorganism)xe2x80x9d by Takeji Hasegawa, edited version, Gakkai Shuppan center, 1985.
Bacillus stearothermophilus JTS 859 (FERM BP-6885)
1. Morphological Characteristics
(1) Shape and size of the cell: Rod, 5.4-6.5 xcexcmxc3x970.7-0.9 xcexcm
(2) Spore: Ellipsoidal-shaped spore is formed. One spore in one cell, the spore being positioned at the end of the cell.
2. Characteristics in Culture
(1) Bouillon liquid culture: culture at 62xc2x0 C. for 2 days
(2) Bouillon agar plate culture: White with slightly yellow tint, glossy, opaque, not piling up. The colony has a wavy circular shape.
(3) Bouillon agar slant culture: White with slightly yellow tint, glossy, opaque, not piling up. Moderate growth.
3. Biochemical Characteristics
Judging from the aforementioned characteristics, the present strain can be determined to be Bacillus stearothermophilus. 
 less than One-pot Reaction greater than 
The method of preparing a glyoxal-guanosine-group compound and the method of preparing a guanosine-group compound of the present invention can be both carried out by a one-pot reaction. Specifically, the initial reaction solution is first prepared by reacting guanosine with a glyoxal aqueous solution. The ribose-group donor (preferably together with a stabilizer), the enzyme solution required for the reaction, and optionally the phosphate ion are added, for the reaction, to the initial reaction solution, whereby a glyoxal-guanosine-group compound can be prepared by a one-pot reaction. In addition, by alkali-decomposing the reaction product, a guanosine-group compound can be prepared by a one-pot reaction.